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. 2016 May 3;6(5):1391-408.
doi: 10.1534/g3.116.027615.

Multiple Targets on the Gln3 Transcription Activator Are Cumulatively Required for Control of Its Cytoplasmic Sequestration

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Free PMC article

Multiple Targets on the Gln3 Transcription Activator Are Cumulatively Required for Control of Its Cytoplasmic Sequestration

Rajendra Rai et al. G3 (Bethesda). .
Free PMC article

Abstract

A remarkable characteristic of nutritional homeostatic mechanisms is the breadth of metabolite concentrations to which they respond, and the resolution of those responses; adequate but rarely excessive. Two general ways of achieving such exquisite control are known: stoichiometric mechanisms where increasing metabolite concentrations elicit proportionally increasing responses, and the actions of multiple independent metabolic signals that cumulatively generate appropriately measured responses. Intracellular localization of the nitrogen-responsive transcription activator, Gln3, responds to four distinct nitrogen environments: nitrogen limitation or short-term starvation, i.e., nitrogen catabolite repression (NCR), long-term starvation, glutamine starvation, and rapamycin inhibition of mTorC1. We have previously identified unique sites in Gln3 required for rapamycin-responsiveness, and Gln3-mTor1 interaction. Alteration of the latter results in loss of about 50% of cytoplasmic Gln3 sequestration. However, except for the Ure2-binding domain, no evidence exists for a Gln3 site responsible for the remaining cytoplasmic Gln3-Myc(13) sequestration in nitrogen excess. Here, we identify a serine/threonine-rich (Gln3477-493) region required for effective cytoplasmic Gln3-Myc(13) sequestration in excess nitrogen. Substitutions of alanine but not aspartate for serines in this peptide partially abolish cytoplasmic Gln3 sequestration. Importantly, these alterations have no effect on the responses of Gln3-Myc(13) to rapamycin, methionine sulfoximine, or limiting nitrogen. However, cytoplasmic Gln3-Myc(13) sequestration is additively, and almost completely, abolished when mutations in the Gln3-Tor1 interaction site are combined with those in Gln3477-493 cytoplasmic sequestration site. These findings clearly demonstrate that multiple individual regulatory pathways cumulatively control cytoplasmic Gln3 sequestration.

Keywords: Gln3; mTorC1; methionine sulfoximine; nitrogen limitation; rapamycin.

Figures

Figure 1
Figure 1
Schematic summarizing nitrogen-responsive regulation of the mTorC1 kinase complex and downstream control of Gln3 localization in nitrogen excess (left pathway) or limitation (right pathway).
Figure 2
Figure 2
Reproducibility of the data in this work. The histograms represent the means of the eight localization experiments contained in this work. They were performed over 4 yr, measuring Gln31-730-Myc13 localization in wild-type JK9-3da transformed with pRR536; error bars represent SD around these means.
Figure 3
Figure 3
Gln3-Myc13 phosphorylation profiles in transformants containing wild type (pRR536) and C-terminal gln3 truncation mutants. Cells were cultured in untreated YNB-glutamine (Gln) or proline (Pro) media; rapamycin (+Rap) was added where indicated. Western blots were performed as described in Materials and Methods. The length of the Gln3 proteins assayed appear above the plasmid numbers. Black dots indicate positions of major species. (A) pRR536, (B) pRR614, (C) pRR622, (D) pRR613, and (E) pRR609.
Figure 4
Figure 4
Plot of the predicted probability of Gln3420–730 being disordered. The cutoff for predictions exhibiting a 5% or less probability of generating a false positive is indicated (line at 0.5). The bold black line indicates the region of Gln3 analyzed in this work.
Figure 5
Figure 5
(A) Region of Gln3 (residues 475–540) that exhibits high homology among 11 strains most related to Saccharomyces cerevisiae. The residue designations are color-coded: red, basic; blue, hydrophobic; green, polar; orange, glycine; yellow, proline and purple, acidic. Boxes beneath the homology series indicate residues shown to be required for cytoplasmic Gln3-Myc13 sequestration, red; a response to rapamycin, black. Substitutions that affected both of these responses are indicated in red-black, and those with very modest effects in light red/pink. (B) Gln3 amino acid substitutions analyzed in this work. Plasmid numbers appear to the left of the sequences. All residues substituted are indicated in pink in the wild type sequence (pRR536) that appears at the top of each panel. Black lines highlight the regions where substitutions were made.
Figure 6
Figure 6
The response of Gln3-Myc13 localization to aspartate substitutions for hydrophobic amino acids in Gln3 region 421 to 439. (A) and (B) Cells were cultured in YNB-glutamine medium (Gln); rapamycin (200 ng/ml) was added where indicated (+ Rap) for 20 min. (C) and (D) Cells were cultured in either YNB-glutamine or proline medium. (E) and (F) Cells were cultured in YNB-ammonia (Am.); Msx (2 mM final concentration) was added for 30 min where indicated (+ Msx). Intracellular Gln3-Myc13 localization was scored as indicated in Materials and Methods as being cytoplasmic (red bars), nuclear-cytoplasmic (yellow bars), or nuclear (green bars). The mutant amino acid substitutions are shown above (A) and in context in Figure 5B.
Figure 7
Figure 7
(A–C) show the response of Gln3-Myc13 localization to substitution of amino acids in Gln3 region 450 to 473. The format of the experiments and presentation of the data were as in Figure 6.
Figure 8
Figure 8
Substitution of alanine for 13 serine residues in the serine/threonine/basic amino acid-rich region of Gln3 largely abolishes cytoplasmic sequestration of Gln3-Myc13 (pRR680) in nitrogen-rich glutamine (A–D) or ammonia (E and F) medium. Aspartate substitutions (pRR682) elicited Gln3-Myc13 intracellular Gln3-Myc13 distributions that were indistinguishable from wild type. The experimental format and presentation of the data were as described in Figure 6.
Figure 9
Figure 9
Substitution of alanine for the N-terminal four serine residues in the serine/threonine/basic amino acid-rich region of Gln3 reduces cytoplasmic sequestration of Gln3-Myc13 (pRR676) in nitrogen-rich glutamine medium (A–D), and largely abolishes it in ammonia medium (E and F). Aspartate substitutions (pRR678) elicited Gln3-Myc13 intracellular Gln3-Myc13 distributions that were indistinguishable from wild type. The experimental format and presentation of the data were as described in Figure 6.
Figure 10
Figure 10
Substitution of alanines for the center eight serine residues in the serine/threonine/basic amino acid-rich region of Gln3 reduces cytoplasmic sequestration of Gln3-Myc13 (pRR1040) in nitrogen-rich glutamine medium (A–D) and largely abolishes it in ammonia medium (E and F). Aspartate substitutions (pRR1043) elicited Gln3-Myc13 intracellular Gln3-Myc13 distributions that were indistinguishable from wild type. The experimental format and presentation of the data were as described in Figure 6.
Figure 11
Figure 11
Effects of serine to aspartate [pRR682 (A) and pRR678 (C)], or alanine [pRR680 (B) and pRR676 (D)], subsitutions on Gln3-Myc13 electrophoretic mobilities in glutamine (Gln)- or proline (Pro)-grown cells, and glutamine-grown cells treated with rapamycin. Black dots indicate positions of the major species.
Figure 12
Figure 12
(A–D) Alanine (pRR1178 and pRR1173) or aspartate (pRR1172 and pRR1180) substitutions for serine residues in the serine/threonine/basic amino acid-rich region of Gln3 with homology to known protein kinase phosphorylation sites. The experimental format and presentation of the data were as described in Figure 6.
Figure 13
Figure 13
Rapamycin sensitivity of wild type and Gln3 amino acid substitution mutants. Transformants were spotted in succeeding 10-fold dilutions on synthetic complete medium, and the same medium containing 50 ng/ml rapamycin. The cells were cultured for just over 4 d at 30° C. The transformation recipient for these experiments was KHC2, a gln3Δ.
Figure 14
Figure 14
Cytoplasmic Gln3-Myc13 sequestration observed in transformants with individual alterations in the Gln3479–493 serine/threonine-rich region (pRR680), and the Gln3-mTor1 interaction site (pRR850, Rai et al. 2013) compared to that which occurs in a transformant containing both sets of alterations (pRR1340). The predicted level of cytoplasmic Gln3-Myc13 sequestration expected if the alterations are additively diminishing cytoplasmic Gln3-Myc13 sequestration are indicated with arrows and yellow lines in the glutamine and ammonia data derived with pRR1340 transformants. The experimental format and presentation of the data were as described in Figure 6.
Figure 15
Figure 15
(A–F)Substitution of glutamate for the hydrophobic (pRR1270) and basic (pRR1272) residues in the proline-rich region between residues required for cytoplasmic Gln3-Myc13 sequestration and the Gln3 response to rapamycin treatment diminishes the rapamycin response. The experimental format and presentation of the data were as described in Figure 6.
Figure 16
Figure 16
Schematic of the C-terminal regulatory region of Gln3. The black bars indicate strong requirements of the residues indicated at the top of the figure for the processes indicated. White regions in or between these bars indicate areas where substitutions had little to no effect. In instances where putative α-helices appear, the hydrophobic and nonhydrophobic residues appear in red balls and green ribbons, respectively (Rai et al. 2013, 2014).

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